Many scientific and commercial methods involve intracellular delivery of materials to the interior of cells. For example, in the medical field, treatment therapies can deliver pharmaceutical agents to the interior of cells. In the field of molecular biology, it is known to deliver genetic material and other biological compounds to the interior of a cell (i.e., transfection). One important application of intracellular delivery of nucleic acids is gene therapy. In addition to gene delivery, pharmaceutical agent delivery compositions can be used to deliver other therapeutic agents, as well as diagnostic and cosmetic agents.
With regard to gene therapy, recent advances in molecular biology have increased the scientific understanding of the genetic basis for disease. Modern tools have provided significant advances in gene therapy. It is now possible to produce nucleotide sequences capable of expressing therapeutic molecules and to package them in expression vectors. In the context of gene therapy, transfection systems are used to deliver the expression vectors to a subject's cells. Once delivered, the nucleic acid is expressed in the target cells to produce proteins and/or peptides. Although gene therapy is promising as a method of treating genetic based diseases, major obstacles remain. Progress in gene therapy has been limited by the lack of gene-delivery systems having a transfection efficiencies which are adequate to deliver therapeutically significant amounts of genetic materials and other pharmaceutical agents into target cells. Furthermore, nucleic acid delivery systems, in particular non-viral systems, have a significant reduction in transfection efficiency in the presence of serum. Success of gene therapy depends on the development of improved transfection systems, with higher transfection efficiency and increased resistance to serum.
Commonly used transfection systems can be grouped into viral and non-viral transfection systems.
2.1 Non-Viral Transfection Systems
Non-viral transfection systems have been explored as delivery mechanisms for genetic materials and other biological compounds. Such non-viral transfection systems include, for example, liposomal systems. A liposome is a compartment bounded by a lipid bilayer. Materials, such as DNA or protein, can be contained within a liposome, either in the liposome compartment, associated with the bilayer, or associated with the liposome exterior, and therapeutic materials can be delivered to the interior of a cell by endosomal uptake or by fusion of the agent-containing liposome with the cell membrane.
Cationic liposomes are the most commonly used non-viral delivery system for both in vitro (1–3) and in vivo (4–6) DNA transfection. A significant problem associated with cationic liposomes is their low transfection efficiency (7,8), both in vitro and in vivo. At least in vivo, this low transfection efficiency has been attributed to inactivation of liposomes by serum (9,10). On the other hand, cationic liposome carriers are relatively easy to produce in large quantities and exhibit minimal toxicity (11). Accordingly, significant effort is presently being devoted to improving the in vivo efficacy of liposomes.
Several recent developments have advanced the prospects for enhanced liposome transfection efficiency. In vivo transfection efficiency of liposomes has been enhanced by the development of new lipids (12) and by the replacement of DOPE (13) with cholesterol, as a helper lipid.
The addition of poly-L-lysine or protamine to cationic liposome carriers is known to enhance the transfection efficiency of liposomes (9,14–17). These highly basic polymers/proteins effectively condense the plasmid DNA, while liposomes neutralize the remainder of the negative charge of the DNA and provide a scaffold for the polymer:DNA complex. Although poly-L-lysine increases transfection of liposome carriers, the addition of serum during transfection markedly reduces the efficiency of this carrier. Thus, poly-L-lysine in combination with liposomes may be somewhat limited as a carrier in the presence of serum.
Lysosomotropic agents have also been used to increase transfection efficiency of liposomes and other cationic carriers. Lysosomotropic agents protect plasmids from hydrolytic digestion within endosomes and/or enable plasmids to escape from endosomes (18–25). These lysosomotropic agents include chloroquine, NH4CL and monesin, which are weak amines that buffer the pre-lysosomal vesicles, and bafilomycin, a specific inhibitor of the vacuolar pump.
Other efforts at improving gene-delivery agents have focused on DNA-condensing and pH buffering properties (26–29). These efforts have yielded dendrimers (Superfect, polyethylenimine) and polyamidoamine whose single repeating subunit contains both a positive charge and buffering capacity. Such polymers have a single repeating subunit with two or more functional features, resulting in an inherent inflexibility, since binding and buffering properties within these polymers are fixed and cannot be varied. It is also likely that the currently available polymers will not be metabolizable, and with prolonged administration, these polymers are likely to be toxic. Previously, polymers with both DNA binding and buffering capacity (e.g. polyamidoamine) in combination with liposomes were found not to enhance transfection efficiency (15). In addition, a large molecular weight histidylated poly-L-lysine polymer has been recently reported in 3 of 4 cell lines to be significantly less effective (between 6 to 21 fold) than polyethylenimine (PEI), a commonly used carrier and one of the more effective carriers in gene transfer systems (30).
There is a need in the art for pharmaceutical agent delivery systems having transfection efficiencies sufficient to deliver therapeutically effective amounts of nucleic acid into cells. There is also a need in the art for carriers that are stable in serum in order for delivery systems to be effective both in vitro and in vivo. Moreover, there is a continuing need in the art for improved non-viral pharmaceutical agent delivery systems capable of delivering pharmaceutical agents into the interior of cells in amounts sufficient to treat conditions such as metabolic and neoplastic conditions in humans.
2.2 Viral Transfection Systems
Various viral transfection systems are also known in the art, such as retroviruses (e.g., murine leukemia virus, avian, lentivirus), adenoviruses and adeno-associated viruses, herpes simplex viruses, rhinovirus, Sendai virus, and Poxviruses. These systems have been proposed for gene therapy. Combinations of viral vectors with non-viral delivery systems have also been utilized to enhance gene delivery (37–44). A significant limitation of viral vectors as transducing agents is their entry into cells. For instance, viral vectors have decreased transduction efficiency and gene expression in a variety of cells such as hematopoeitic stem cells. Alternatively, in the case of retroviruses, specific envelop proteins limit the entry of the retrovirus into cells and consequently, the transduction (transfection) efficiency in these cell lines is markedly reduced. There is a need in the art to enhance the entry of viral vectors into cells.